Isolation,Purification And Identification Of Antimicrobial Lipopeptide From Marine Bacillus Subtilis

Bacillus subtilis is a widely used probiotics which metabolize low molecular antimicrobial peptides.Lipopeptide is a kind of green,safe and efficient alternatives to antibiotics.It has property of low resistance,low toxicity,broad antibacterial spectrum,high thermal stability,biodegradable and also known as lipopeptide antibiotics.In recent years,secondary metabolites were found in marine microbes which have the physiological significance.These substances usually have a new unique structure that have a good performance in terms of resistance to bacteria,antineoplasticity,antivirus.In this study,the lipopeptides produced by Bacillus subtilis strain HS-A38 isolated from the intestinal tract of sea cucumber.The lipopeptides were subjected to gene detection,component analysis,isolation,purification and activity.Four of antimicrobial peptides genes were detected from HS-A38 genomic DNA by PCR,and the target genes conding Bacilysin,Iturin,Bacillomycin and Fengycin were analysed.The crude extract of lipopeptide LP from the Bacillus subtilis HS-A38 fermentation broth was purified by acid precipitation and organic solvent extraction.Its yield is 1.955 g/L.The components of the crude extract of the lipopeptides were determined by thin layer chromatography(TLC).Four components LP1,LP2,LP3 and LP4 were found.By using reversed-phase high-performance liquid chromatography(RP-HPLC),it was found that the liquid purification peak recovery of LP were obtained three purified groups,that is the peak A,peak B and peak C as the retention time was 25?28 min,28?32 min and 33?39 min.Furthermore,the ultra-high performance liquid phase and ion trap tandem mass spectrometry(UPLC-LTQ Orbitrap)analysis was performed on the three groups.The main components of the two peaks of A and B were Bacillomycin,whereras the group of peak C had components of the complex composition including Bacilysin,Fengycin and Iturin.LP1,LP2,LP3,and LP4 were isolated from the preparation of chromatographic plates in order to isolate single-component and broad-spectrum bacteriostatic Bacillomycin,and the antimicrobial activity was tracked.Preparative plate separation result showed that component LP3 had a high inhibitory effect on four bacteria and a fungal yeast.The crude extract LP was isolated to component LP3 in the conditions that chloroform and methanol ratio is 7:3.LP3 was purified by reversed phase high performance liquid chromatography(RP-HPLC)and matrix assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS).The results showed that LP3 had a single cluster LP3-1,at a retention time of 25 to 28 min.Its purity is 85.24%.MALDI-TOF-MS results analysis and data comparison shows that component LP3-1 in the main component was Bacillomycin D.Isolation,extraction and application of Bacillomycin D will provide protection for the prevention of bacterial and fungal diseases in aquatic industry.